The diseases caused by retroviruses (e.g., AIDS and cancer) cannot be cured and have no vaccines for prevention. Therefore, a better understanding of the viruses that cause these diseases is needed. One of the least understood parts of the retrovirus replication cycle is budding, the step during which new virus particles emerge from the infected-cell surface and are release to spread the infection to new cells. Budding is mediated by the Gag protein, and during previous funding periods we have identified and characterized the primary domains (the M, I, and L domains) of this protein that are needed for particle released from the plasma membrane. Although a good understanding of Gag proteins has been acquired, the contributions of the host cell to the budding process are a mystery. The long-range goal of this Continuation Application is to identify and characterize the host factors required for budding. This is a challenging endeavor, but fortunately, glimpses of the host machinery have been made during the previous funding period. Interestingly, all the evidence points towards the involvement of components of the endocytic and vesicular trafficking machinery of the cell. We request funding to pursue two large Specific Aims. The first is to thoroughly analyze the role of ubiquitin in retrovirus budding. During the previous funding period, we discovered that this host protein is actually part of the budding machinery at the plasma membrane. This raises a variety of important clues and questions that need to be addressed. The second aim is to identify and characterize additional host proteins that are involved in budding. Again, clues from our previous work provide guidance on how to do this. For both aims, the primary focus will be Rous sarcoma virus, but as in the previous funding periods, we will utilize other enveloped viruses when they offer the possibility of gaining additional insights. Indeed, we have shown that the mechanism of budding used by retroviruses has features in common with unrelated viruses, and hence what we learn from these studies is likely to be useful for a variety of other viral diseases as well.